This is a competing renewal of an ROl grant with the long term goal of understanding how Epstein-Barr virus (EBV) gene expression is regulated during immortalizing latency. EBV establishes a life-long infection within the infected host, and is closely associated with the development of endemic Burkitt's lymphoma, nasopharyngeal carcinoma, 30-50 percent of Hodgkin's disease, and nearly half of the lymphomas that arise in immunosuppressed patients. Notably, EBV infection of peripheral resting B cells results in growth transformation resulting, ex vivo, in the generation of immortalized lymphoblastoid cell lines. Based on recent analyses of EBV infection in seropositive individuals, it seems likely that the ability of EBV to drive B cell proliferation, and subsequent differentiation, plays an important role in the dissemination of virus infected B cells and the establishment of a long-lived latency reservoir in memory B cells (in which there is very limited viral gene expression). Understanding how EBV regulates viral gene expression during immortalizing latency may ultimate provide strategies for interfering with this phase of the virus life cycle, which could interfere with the establishment of latency in the memory B cell compartment. Within the scope of this proposal, we will continue to focus our analyses on identifying and characterizing cis-elements involved in regulated EBNA gene expression during the immortalizing latency program of EBV. Aim 1. Generation of a cottontop marmoset LCL immortalized with a packaging defective and replication null EBV to serve as an inducible reservoir for non-immortalizing EBV mutants. Aim 2. Generation and characterization of EBV harboring mutations in cis-elements thought to be involved in regulating EBNA gene transcription in EBV immortalized lymphoblastoid cell lines. Aim 3. Analysis of the requirements for Wp activity during initial stages of infection of primary B cells. Aim 4. Analysis of Wp methylation during the establishment and maintenance of immortalizing latency in B cells - role of methylation in regulating Wp activity in lymphoblastoid cell lines.